Composition, structure and interactions of the Cleavage and Polyadenylation Specificity Factor (CPSF) involved in mammalian mRNA 3' end processing

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The ends of eukaryotic mRNAs are generated by a processing reaction in which the mRNA precursor is first shortened by endonucleolytic cleavage and the upstream cleavage fragment is then polyadenylated. Within a large assembly of proteins required for the processing reaction, the heterooligomeric cleavage and polyadenylation specificity factor (CPSF) is considered the central factor: CPSF is involved both in cleavage and polyadenylation, it contains the enzyme catalyzing pre-mRNA cleavage, it recognizes the AAUAAA polyadenylation signal, and it endows poly(A) polymerase with specificity for an AAUAAA-containing substrate RNA. However, neither the composition of CPSF nor its molecular interactions are well understood.We have reconstituted CPSF from six overexpressed subunits.The reconstituted factor is active in polyadenylation. Two of the six subunits are dispensable for this reaction, but are probably essential for pre-mRNA cleavage. As the two other proteins required for polyadenylation, poly(A) polymerase and the nuclear poly(A) binding protein, can be produced in E. coli, a fully reconstituted reaction is now available for mechanistic studies.The composition of the minimal CPSF active in AAUAAA-dependent polyadenylation will be determined with respect to types of subunits and the stoichiometry of their association.The RNA binding specificity of CPSF and the contributions of individual subunits to RNA recognition will be studied. This concerns both the AAUAAA polyadenylation signal and additional sequences possibly recognized by CPSF.The interactions of CPSF subunits with each other and, most importantly, with poly(A) polymerase will be determined. We will ask whether the interaction with poly(A) polymerase is regulated by the length of the poly(A) tail, as postulated in a published model for poly(A) tail length control. We will investigate how CPSF stimulates poly(A) polymerase and whether two other processing factors, cleavage factor I and cleavage stimulation factor, both available in overproduced and purified form, can indeed stimulate polyadenylation in a reconstituted system as has been reported for a less purified system.Symplekin and cleavage factor II will be overexpressed to complete the set of known 3 prime processing factors, and a reconstitution of the cleavage reaction from overproduced and purified proteins will be attempted. If this is not successful, identification of the missing factor(s) will be attempted.Finally, we will collaborate with structural biologists to help elucidate the structure of CPSF by means of cryo-electron microscopy and X-ray crystallography